Voltage-sensitive potassium (Kv) channels are proteins that exist in the membranes of all electrically excitable cell types. Kv1.4 (the mammalian homologue of Shaker) and all Kv4 (Shal-type) subunits generate potassium-selective current phenotypes designated "Ito" in cardiac myocytes and "IA" in neurons. Due to their rapid activation and subsequent inactivation kinetics, both channel types can significantly modulate action potential repolarization and frequency-dependent electrical signaling. IA/Ito phenotypes have thus been hypothesized to play important functional roles in both the cardiovascular and nervous systems. In particular, Kv4 channels have recently been importantly implicated in neural mechanisms underlying both memory and pain perception. While much is understood about molecular mechanisms underlying activation, inactivation, and recovery in Shaker/Kv1.4, the corresponding mechanisms in Kv4 are presently undetermined. Nonetheless, there is general consensus that "conventional" Shaker N- and C- type inactivation mechanisms are not involved. Kv4 channels also display prominent inactivation from pre-activated closed states (closed state inactivation or CSI), a process which is not significant in Shaker. Therefore, as an alternative to the conventional Shaker/Kv1.4 gating model, I hypothesize that transitions in the Kv4 voltage sensing domain (VSD), and in particular those mediated by S4 positively charged arginine (R) residues, are primarily responsible for regulating not only activation and deactivation, but also CSI and recovery. CSI thus either possesses inherent voltage dependence or is coupled to activation by a mechanism significantly different from that in Shaker. As a result, Kv4 recovery (from both open inactivated and closed inactivated states) will be coupled to deactivation. Using Xenopus laeavis oocytes as an expression system, a combination of mutagenic and functional kinetic analysis (two microelectrode voltage clamp, cut open oocyte voltage clamp) will be employed to gain initial insights into molecular and biophysical mechanisms underlying CSI and recovery of Kv4.3 subunits. The Specific Aim will be to develop novel methods for measurement of Kv4.3 subunit gating currents "Ig" and to determine how various S4 arginine (R) mutants alter them. PUBLIC HEALTH RELEVANCE: Kv4 channels are important for normal functioning of the heart, and are believed to be directly involved in several functions/dysfunctions of the nervous system, including learning and memory, epilepsy, and pain perception. The proposed research will provide new molecular and biophysical insights into basic mechanisms regulating Kv4 channels, and may thus provide the basis for development of more effective therapeutic treatments.